STEROID-BINDING AND DIMERIZATION DOMAINS OF HUMAN SEX HORMONE-BINDING GLOBULIN PARTIALLY OVERLAP - STEROIDS AND CA2+ STABILIZE DIMER FORMATION

Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with a single steroid-binding site for biologically active sex steroids, and a methionine at position 139 (M139) interacts with the photoaffinity ligand, Delta(6)-testosterone. We have introduced amino acid substitutions into this and other locations in the SHBG molecule and have examined their impact on steroid binding and dimerization. As a result, substitutions at residues 134-139 generate alterations in steroid-binding specificity. In particular, substitutions at residues 134-138 were characterized by altered binding affinities for estradiol relative to 5 alpha-dihydrotestosterone (DHT), and one of them (R135L) also showed a 2-fold increase in affinity for C19 steroids with a 3 beta-hydroxy group. Unlike all of the other mutants we have examined, the M139W mutant has a 5-fold lower affinity for DHT, and its affinities for testosterone, 5 alpha-androstane-3 beta,17 beta-diol, and estradiol also appear to be reduced to a similar extent. By contrast, M139W appears to bind androst-5-ene-3 beta,17 beta-diol with only 2-fold less affinity than wild-type SHBG, while its affinity for 19-nortestosterone remains unaffected. Substitutions at other positions, including those immediately C-terminal to M139, had no effect on steroid-binding affinity and/or specificity. These data provide evidence that residues 134-139 influence the recognition of specific A/B ring conformations of steroid ligands and may constitute part of the steroid-binding domain. We have also found that substitutions at residues 138-148 impair dimerization and that this defect may be abrogated by occupancy of the steroid-binding site. The removal of divalent cations also destabilizes dimer formation of mutants with substitutions at residues 140-148, and Ca2+ or Zn2+, but not Mg2+, can restore their ability to form dimers. Steroid ligands and divalent cations also appear to act independently to stabilize dimer formation. These data lead us to conclude that the steroid-binding and dimerization domains of human SHBG partially overlap and that steroid ligands and divalent cations influence the dimerization interface to enhance subunit association.